In eukaryotic cells, vesicle trafficking is the principle mechanism by which materials are transported between membrane bound compartments or to the plasma membrane, and it is tightly regulated to ensure that cargo is delivered to the correct destination at the correct time. Our effort is directed at understanding the molecular basis for this regulation, and we propose to investigate instances of both spatial and temporal regulation, focusing on events as the vesicle arrives at the acceptor compartment. We bring to bear structural biology, biochemical and biophysical approaches. In Aim 1 we investigate how guanine nucleotide exchange factors (GEFs) recognize and activate members of the Rab family of small GTPases, which play a key role in defining organelle identity and hence in ensuring correct cargo delivery. We focus on how the DENN-domain proteins, a major family of GEFs in higher eukaryotes, interact with their Rab partners. The crystal structure determination of a DENN-domain/Rab complex is well underway and, together with kinetic and thermodynamic studies, will elucidate recognition and activation mechanisms. In Aim 2, we study how complexes in the TRAPP family, tethering factors that act in vesicle recognition at the acceptor compartment, are localized to different compartments as different subunits are added to a shared core. An important aspect of this work is the determination of the structure for TRAPPIII or a TRAPPIII subcomplex. Intact TRAPPIII as well as key subcomplexes have been reconstituted. Low resolution electron microscopy reconstructions have been obtained for the intact complex and initial crystallization conditions identified for a subcomplex. Lastly, in Aim 3 we examine how specialized proteins in nerve cells regulate the assembly of SNARE complexes, which drive vesicle fusion and cargo delivery, so that neurotransmitter is released only in response to an action potential. We will determine how the synaptic proteins complexin and synaptotagmin regulate SNARE assembly at the plasma membrane. We have determined a crystal structure of complexin bound to a mimetic of a pre-fusion SNARE complex that explains how SNARE assembly can be clamped, pending an action potential, and propose further studies aimed at understanding clamping and clamp release. PUBLIC HEALTH RELEVANCE: Membrane traffic is a fundamental biological process for organelle formation, nutrient uptake, and the secretion of hormones and neurotransmitters. It is central to release in many areas of endocrine and exocrine physiology, and imbalances in these processes give rise to more than 50 human diseases, including diabetes. Additionally, many pathogens hijack membrane traffic mechanisms in order to proliferate and ensure their survival, so that an understanding of the basic mechanisms underlying membrane traffic is essential for protecting against these pathogens.